Recombinant micelle and method of in vivo assembly

ABSTRACT

A method of in vivo assembly of a recombinant micelle including: introducing a plasmid into a plant cell, wherein: the plasmid includes a segment of deoxyribonucleic acid (DNA) for encoding a ribonucleic acid (RNA) for a protein in a casein micelle, the segment of DNA is transcribed and translated; forming recombinant casein proteins in the plant cell, wherein: the recombinant casein proteins include a κ-casein and at least one of an αS 1 -casein, an αS 2 -casein, a β-casein; and assembling in vivo a recombinant micelle within the plant cell, wherein: an outer layer of the recombinant micelle is enriched with the κ-casein, an inner matrix of the recombinant micelle include at least one of the αS 1 -casein, the αS 2 -casein, the β-casein.

SEQUENCE LISTING

The content of the ASCII text file of the sequence listing named “Mozza62162-701_301_Sequence_Listing.txt”, which is 30,917 bytes in size wascreated on Apr. 6, 2022 and electronically submitted via EFS-Web on Apr.8, 2022, is incorporated herein by reference in its entirety.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/741,680, filed Jan. 13, 2020, which claims the benefit of U.S.Provisional Patent Application No. 62/939,247, filed Nov. 22, 2019, allof which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

An embodiment of the present disclosure relates generally to a micelleand more particularly to recombinant micelle and method of in vivoassembly in a plant cell.

BACKGROUND

Casein micelles account for more than 80% of the protein in bovine milkand are a key component of all dairy cheeses. Casein micelles includeindividual casein proteins are produced in the mammary glands of bovinesand other ruminants. The industrial scale production of the milk that isprocessed to yield these casein micelles, primarily in the form of curdsfor cheese production, typically occurs on large-scale dairy farms andis often inefficient, damaging to the environment, and harmful to theanimals. Dairy cows contribute substantially to greenhouse gasses,consume significantly more water than the milk they produce, andcommonly suffer from dehorning, disbudding, mastitis, routine forcedinsemination, and bobby calf slaughter.

Accordingly, there is a need for an in vivo plant-based caseinexpression system which allows for purification of biologically activecasein proteins that is cost effective at industrial scale.

Solutions to these problems have been long sought but prior developmentshave not taught or suggested any solutions and, thus, solutions to theseproblems have long eluded those skilled in the art.

DISCLOSURE OF INVENTION

An embodiment of the present invention provides a method of in vivoassembly of a recombinant micelle including: introducing a plasmid intoa plant cell, wherein: the plasmid includes a segment ofdeoxyribonucleic acid (DNA) for encoding a ribonucleic acid (RNA) for aprotein in a casein micelle, the segment of DNA is transcribed andtranslated; forming recombinant casein proteins in the plant cell,wherein: the recombinant casein proteins include a κ-casein and at leastone of an αS₁-casein, an αS₂-casein, a β-casein; and assembling in vivoa recombinant micelle within the plant cell, wherein: an outer layer ofthe recombinant micelle is enriched with the κ-casein, an inner matrixof the recombinant micelle include at least one of the αS₁-casein, theαS₁-casein, the β-casein.

An embodiment of the present invention provides a recombinant micelleincluding: an outer layer enriched with a κ-casein; and an inner matrixincluding at least one of a αS₁-casein, a αS₁-casein, a β-casein.

An embodiment of the present invention provides a plasmid including asegment of deoxyribonucleic acid (DNA) for encoding a protein in acasein micelle wherein the segment of DNA includes a promoter and aN-terminal signal peptide.

Certain embodiments of the disclosure have other steps or elements inaddition to or in place of those mentioned above. The steps or elementswill become apparent to those skilled in the art from a reading of thefollowing detailed description when taken with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example of a flow for forming in vivo casein micelles in anembodiment.

FIG. 2 is an example of a schematic illustration of a plasmid used inFIG. 1.

FIG. 3A is an example with additional details from the planttransformation to the post-translation modification.

FIG. 3B is an example with additional details for the in vivo formation.

FIG. 3C is an example of a schematic illustration of a transcription ofproteins which impart herbicide resistance to the transformed plant.

FIG. 3D is an example of a schematic illustration of suppression ofnative seed storage proteins by RNAi transcribed by a portion of theplasmid of FIG. 1.

FIG. 3E is an example of a schematic illustration of a transcription ofa portion of the plasmid of FIG. 1 and resulting proteins used toincrease calcium concentrations in the plant cell.

FIG. 3F is an example of a schematic illustration of a transcription ofa portion of the plasmid of FIG. 1 and resulting proteins used toincrease phosphate concentrations in the plant cell.

FIG. 3G is an example with further additional details of the in vivoformation.

FIG. 4 is an example of a schematic illustration of a portion of aplasmid in Arabidopsis.

FIG. 5 is an example of a schematic illustration of a portion of aplasmid in Arabidopsis for a screenable marker in plants.

FIG. 6 is an example of a schematic illustration of a portion of aplasmid in soybean.

FIG. 7 is an example of a schematic illustration of a portion of aplasmid in soybean for herbicide resistance in plants.

FIG. 8 is an example of a schematic illustration of a portion of theplasmid of FIG. 1 for soybean for suppression of native seed storageproteins in plants.

FIG. 9 is an example of a schematic illustration of a portion of aplasmid for soybean to regulate intracellular concentrations of mineralswhich can enhance micelle formation.

FIG. 10 is an example of a flow for the purification of micelles formedin vivo in soybean.

DETAILED DESCRIPTION

The following embodiments are described in sufficient detail to enablethose skilled in the art to make and use the invention. It is to beunderstood that other embodiments would be evident based on the presentdisclosure, and that system, process, or mechanical changes may be madewithout departing from the scope of an embodiment of the presentdisclosure.

In the following description, numerous specific details are given toprovide a thorough understanding of the invention. However, it will beapparent that the invention may be practiced without these specificdetails. In order to avoid obscuring an embodiment of the presentdisclosure, some well-known techniques, system configurations, andprocess steps are not disclosed in detail.

The drawings showing embodiments of the system are semi-diagrammatic,and not to scale and, particularly, some of the dimensions are for theclarity of presentation and are shown exaggerated in the drawingfigures. Similarly, although the views in the drawings for ease ofdescription generally show similar orientations, this depiction in thefigures is arbitrary for the most part. Generally, the invention can beoperated in any orientation.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

Referring now to FIG. 1, therein is shown an example of a flow forforming in vivo casein micelles in an embodiment. In this example, FIG.1 depicts the flow for forming the casein micelles by a planttransformation, a recombinant casein protein formation, apost-translation modification, and an in-vivo formation. As a specificexample, FIG. 1 is a schematic illustration of the elements of a plasmidof this embodiment and its use in creation of micelles in vivo in aplant cell.

In this example for the plant transformation, a plant is transformedusing a plasmid including a single transcription unit set. As usedherein “plasmid” is a deoxyribonucleic acid (DNA) molecule capable ofreplication in a host cell and to which another DNA segment can beoperatively linked so as to bring about replication of the attached DNAsegment. As it relates to this example, methods for plant transformationinclude microprojectile bombardment as illustrated in U.S. Pat. Nos.5,015,580; 5,550,318; 5,538,880; 6,153,812; 6,160,208; 6,288,312 and6,399,861, all of which are incorporated herein by reference. Methodsfor plant transformation also include Agrobacterium-mediatedtransformation as illustrated in U.S. Pat. Nos. 5,159,135; 5,824,877;5,591,616 and 6,384,301, all of which are incorporated herein byreference. Recipient cells for the plant transformation include, but arenot limited to, meristem cells, callus, immature embryos, hypocotylsexplants, cotyledon explants, leaf explants, and gametic cells such asmicrospores, pollen, sperm and egg cells, and any cell from which afertile plant may be regenerated, as described in U.S. Pat. Nos.6,194,636; 6,232,526; 6,541,682 and 6,603,061 and U.S. PatentApplication publication US 2004/0216189 A1, all of which areincorporated herein by reference.

Continuing this example for the plant transformation, the plasmidincluding the single transcription unit set is shown and abbreviated inFIG. 1 as 1-TUS PLASMID. The transcription unit set included on thisplasmid is transcription unit set 1 shown and abbreviated in FIG. 1 asTUS₁. As used herein “transcription unit set” is a segment of DNAincluding one or more transcription units. The purpose of atranscription unit set includes but is not limited to proteinexpression, gene suppression, regulatory ribonucleic acid (RNA)production, and herbicide resistance. As used herein “transcriptionunit” is a segment of DNA including at least a promoter DNA andtranscribable DNA. As used herein “promoter” means regulatory DNA forinitiating RNA transcription. A “plant promoter” is a promoter capableof initiating transcription in plant cells whether or not its origin isa plant cell. As used herein “terminator” means any DNA sequence thatcauses RNA transcription to terminate.

Further continuing this example for the plant transformation shown inFIG. 1 as an embodiment, the transcription unit set 1 includes foursegments of DNA; each encoding RNA for one of the four proteins found ina casein micelle: an αS₁ casein, an αS₂ casein, a β casein, and a κcasein. For clarity and as an example, the genes encoding the αS₁casein, the αS₂ casein, the β casein, and the κ casein are shown andabbreviated in FIG. 1 as αS₁, as αS₂, as (3, and as κ, respectively, andshown and annotated in FIG. 1 as MICELLAR PROTEIN GENE. Each DNA segmentencoding RNA for one of the four proteins found in a casein micelle isoperably linked to a promoter, shown and abbreviated in FIG. 1 as P, andincludes a plant-derived, tissue specific, N-terminal signal peptide,shown and abbreviated in FIG. 1 as SP. As used herein “operably linked”is the association of two or more DNA fragments in a DNA construct suchthat the function of one is controlled by the other, for example DNAencoding a protein associated with DNA encoding a promoter. In someembodiments, the N-terminal signal peptide targets the recombinantcasein proteins to the plant vacuoles. In other embodiments, therecombinant casein proteins are targeted to and retained in theendoplasmic reticulum.

As an example for the recombinant casein protein formation, when thefour segments of DNA included in transcription unit set 1 aretranscribed and translated in a transgenic plant (not shown), fourrecombinant casein proteins, each including a plant-derived tissuespecific signal peptide, are formed in the cytoplasm of the plant cell.The recombinant casein proteins are shown and abbreviated in FIG. 1 asαS₁-CASEIN, as αS₂-CASEIN, as β-CASEIN, and as κ-CASEIN, respectively,and are also referred to herein as “recombinant casein proteins” forbrevity. As used herein, “transgenic” plant is a plant whose genome hasbeen altered by the stable integration of recombinant DNA. As an exampleof stable integration, the transgenic plant includes a plant regeneratedfrom an originally-transformed plant cell and progeny transgenic plantsfrom later generations or crosses of a transformed plant. As used herein“recombinant DNA” refers to DNA which has been synthesized, assembled orconstructed outside of a cell. Examples of recombinant DNA can includeDNA containing naturally occurring DNA or complementary DNA (cDNA) orsynthetic DNA.

As it relates to this example for the post-translation modificationshown in FIG. 1 as an embodiment, the four recombinant casein proteinsin the cytoplasm of the plant cell include the αS₁-casein, theαS₂-casein, the β-casein, and the κ-casein, each including a signalpeptide (SP) that localizes the recombinant casein protein to specificorganelles, for example the secretory pathway and protein storagevacuoles, in the plant cell. The signal peptide is removed from therecombinant casein proteins during post-translational modification thatoccurs in the endoplasmic reticulum and the Golgi apparatus of the plantcell. For clarity in this example, the endoplasmic reticulum and theGolgi apparatus are shown and abbreviated in FIG. 1 as ER, and as GOLGI,respectively. In this embodiment and example, phosphorylation occurs onthe recombinant casein proteins prior to, during, or after migration toa specific tissue, shown in FIG. 1 as circles enclosing the letter “P”attached to each of the recombinant casein proteins. In otherembodiments and examples, one or more post-translational modificationsof the recombinant casein proteins can occur, including phosphorylation,glycosylation, ubiquitination, nitrosylation, methylation, acetylation,lipidation and proteolysis. In other embodiments no post-translationalmodifications occur on the recombinant casein proteins, or in otherwords, the post-translation modification is optional.

Continuing this example for the in vivo formation as an embodiment, anouter layer of the micelle is enriched in recombinant κ-casein shown andabbreviated in FIG. 1 as κ, and an inner matrix of the micelle includesthe recombinant αS₁-casein, the recombinant αS₂-casein, the recombinantβ-casein, the calcium and the phosphate, shown in and annotated in FIG.1 as αS₁, αS₂, as αS, and β, respectively. Micelle formation is enhancedby the presence of intracellular calcium and phosphate, shown andabbreviated in FIG. 1 as Ca and P, respectively.

Referring now to FIG. 2, therein is shown an example of a schematicillustration of a plasmid used in FIG. 1. As a specific example, FIG. 2is a schematic illustration of the elements of a plasmid of thisembodiment.

In this example for the plant transformation of FIG. 1, an embodimentprovides a plant that is transformed with one or more transfer DNAsincluding one or more transcription unit sets. As used herein “transferDNA” (T-DNA) is DNA which integrates or is integrated into a genome.

For example, an Agrobacterium-mediated transformation T-DNA is part of abinary plasmid, which is flanked by T-DNA borders, and the binaryplasmid is transferred into an Agrobacterium tumefaciens strain carryinga disarmed tumor inducing plasmid. Also for example, for a biolisticmediated transformation a gene gun is used for delivery of T-DNA, whichis typically a biolistic construct containing promoter and terminatorsequences, reporter genes, and border sequences or signaling peptides,to cells.

Continuing the example of a T-DNA used to transform a plant in anembodiment, the T-DNA includes four transcription unit sets: atranscription unit set 1, a transcription unit set 2, a transcriptionunit set 3, and a transcription unit set 4. For clarity, thetranscription unit set 1, the transcription unit set 2, thetranscription unit set 3, and the transcription unit set 4 are shown andabbreviated in FIG. 2 as TUS₁, as TUS₂, as TUS₃, and as TUS₄,respectively.

In this example as an embodiment, TUS₁ includes one transcription unitfor each of the four casein proteins found in a casein micelle of FIG.2: a transcription unit 1-1 includes DNA encoding αS₁-casein, atranscription unit 1-2 includes DNA encoding β-casein, a transcriptionunit 1-3 includes DNA encoding κ-casein, and a transcription unit 1-4includes DNA encoding αS₁-casein. For clarity and brevity, thetranscription unit 1-1, the transcription unit 1-2, the transcriptionunit 1-3, and the transcription unit 1-4 are shown and abbreviated inFIG. 2 as TU₁₋₁, as TU₁₋₂, as TU₁₋₃, and as TU₁₋₄, respectively. Eachtranscription unit in TUS₁ can also include DNA encoding the sameplant-derived signal peptide. Additionally, each transcription unit inTUS₁ includes a promoter and a transcriptional terminator.

Continuing this example as an embodiment, TUS₂ includes onetranscription unit, shown and abbreviated in FIG. 2 as TU₂₋₁, thatincludes a promoter, DNA encoding phosphinothricin acetyltransferase,and a transcriptional terminator. In other embodiments, TUS₂ can includeone or more genes encoding a selectable marker that can impart herbicideor antibiotic resistance which enables the selection of transformedplants that produce micelles in vivo. Genes enabling selection oftransformed plants include those conferring resistance to antibiotics,including as examples kanamycin, hygromycin B, gentamicin, andbleomycin. Genes enabling selection of transformed plants also includethose conferring resistance to herbicides, including as examples aglyphosate herbicide, a phosphinothricin herbicide, an oxynil herbicide,an imidazolinone herbicide, a dinitroaniline herbicide, a pyridineherbicide, a sulfonylurea herbicide, a bialaphos herbicide, asulfonamide herbicide, and a glufosinate herbicide. Examples of suchselectable markers are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047, all of which are incorporated hereinby reference. In other embodiments, TUS₂ includes one or more genesexpressing a screenable marker which enables the visual identificationof transformed plants that produce micelles in vivo. Genes expressing ascreenable marker include genes encoding a colored or fluorescentprotein, including as examples luciferase or green fluorescent protein(U.S. Pat. No. 5,491,084, herein incorporated by reference), and genesexpressing β-glucuronidase or uidA gene (U.S. Pat. No. 5,599,670, hereinincorporated by reference) for which various chromogenic substrates areknown. In some embodiments, each of the genes encoding a selectable orscreenable marker are operably linked to an inducible promoter, such asfor example a NOS promoter, or a tissue-specific promoter, such as forexample a promoter from the soybean a′ subunit of β-conglycinin, suchthat the translation of the selectable or screenable markers can beregulated.

Continuing this example as an embodiment, TUS₃ includes twotranscription units that yield untranslated RNA molecules that suppressnative seed protein gene translation. The first transcription unit inTUS₃, a transcription unit 3-1, includes the sense strand, or codingstrand, of DNA encoding soybean Glycinin1, and the antisense strand, ornon-coding strand, of DNA encoding soybean Glycinin1 separated by thepotato IV2 intron. For clarity and brevity, the transcription unit 3-1the sense strand or coding strand of DNA encoding soybean Glycinin1, andthe antisense strand or non-coding strand of DNA encoding soybeanGlycinin1, the potato IV2 intron are shown and annotated in FIG. 2 asTU₃₋₁ as GY1 SENSE, as GY1 ANTISENSE, and as IV2 INTRON, respectively.The second transcription unit in TUS₃, a transcription unit 3-2 includesthe sense strand, or coding strand, of DNA encoding β-conglycinin 1 andthe antisense strand, or non-coding strand, of DNA encoding(3-conglycinin 1 separated by the potato IV2 intron. For clarity andbrevity, the transcription unit 3-2, the sense strand or coding strandof DNA encoding β-conglycinin 1, the antisense strand or non-codingstrand of DNA encoding β-conglycinin 1, and the potato IV2 intron areshown and annotated in FIG. 2 as TU₃₋₂ as CG1 SENSE, as CG1 ANTISENSE,and as IV2 INTRON, respectively.

In other embodiments, TUS₃ includes other transcription units that yielduntranslated RNA molecules that suppress native seed protein genetranslation. As an example, in other embodiments, TUS₃ includes onetranscription unit, a transcription unit 3-1, that includes a promoterfrom the soybean GY4 gene (SEQ ID NO:15), a miR319a microRNA fromArabidopsis thaliana that has been modified such that the homologousarms of the microRNA hairpin contain 21 nucleotide sequences matching aportion of the soybean GY1 gene sequence (SEQ ID NO:10), and a NOStranscriptional terminator (SEQ ID NO:35) (not shown).

Continuing this example as an embodiment, TUS₄ includes twotranscription units that encode proteins which alter the intracellularenvironment in a manner that optimizes the production of micelles havingrequisite attributes including size, mineral content, protein content,protein distribution, and mass. The first transcription unit in TUS₄, atranscription unit 4-1 includes a promoter, DNA encoding oxalatedecarboxylase, and a transcriptional terminator. For clarity andbrevity, the transcription unit 4-1 is shown and abbreviated in FIG. 2as TU₄₋₁. The second transcription unit in TUS₄, a transcription unit4-2, includes a promoter, DNA encoding phytase, and a transcriptionalterminator. For clarity and brevity, the transcription unit 4-2 is shownand abbreviated as TU₄₋₂. In this embodiment, transcription andtranslation of TU₄₋₁ yields an oxalate-degrading enzyme which increasesthe amount of free intracellular calcium available for capture andinclusion during micelle formation. Also in this embodiment,transcription and translation of TU₄₋₂ yields a phytase enzyme whichincreases the amount of free intracellular phosphate available forcapture and inclusion during micelle formation. In some embodiments,each of the genes encoding oxalate-degrading enzymes or phytase enzymesare operably linked to a constitutive promoter, tissue specific promoteror an inducible promoter, such as for example, a nopaline synthasepromoter or a promoter from the soybean β-conglycinin gene, such thatthe translation of proteins which alter the intracellular environmentcan be regulated. In some embodiments, TUS₄ includes both atranscription unit 4-1 that increases the intracellular calciumconcentration and a transcription unit 4-2 that increases theintracellular phosphate concentration. In other embodiments, TUS₄includes only a transcription unit 4-1 that increases the intracellularcalcium concentration. In other embodiments, TUS₄ includes only atranscription unit 4-2 that increases the intracellular phosphateconcentration.

In other embodiments, TUS₄ includes transcription units that increasethe intracellular calcium concentration by expressing an oxalate oxidaseenzyme (not shown). As an example, in other embodiments, TUS₄ includesone transcription unit, a transcription unit 4-1, that includes apromoter from the soybean GY4 gene (SEQ ID NO:15), the coding sequencefor the oxalate oxidase 1 coding sequence from wheat that has been codonoptimized for expression in soybean (SEQ ID NO:9), and the NOStranscriptional terminator (SEQ ID NO:35) (not shown). In otherembodiments, TUS₄ includes transcription units that increase theintracellular phosphate concentration by suppressing the expression ofthe soybean myo-inositol-3-phosphate synthase (MIPS1) gene. As anexample, in other embodiments, TUS₄ includes one transcription unit, atranscription unit 4-2, that includes a promoter from the soybean GY4gene (SEQ ID NO:15), a portion of the MIPS1 coding sequence lacking astart codon (SEQ ID NO:21), the IV2 intron from potato (SEQ ID NO:25),the antisense of the MIPS1 sequence (SEQ ID NO:22), and the NOStranscriptional terminator (SEQ ID NO:35) (not shown).

In some embodiments of the disclosure, transcription unit sets areassembled in numeric order. In other embodiments, transcription unitsets can be assembled in any order.

In some embodiments of the disclosure, the plant is transformed with aplasmid that contains transcription unit sets TUS₁, TUS₂, TUS₃, andTUS₄. In other embodiments of the disclosure, the plant is transformedwith a plasmid that contains only transcription unit set TUS₁.

In some embodiments of the disclosure, the plant is transformed with aplasmid that contains transcription unit sets TUS₁, and TUS₂. In otherembodiments of the disclosure, the plant is transformed with a plasmidthat contains transcription unit sets TUS₁, TUS₂, and TUS₃. In otherembodiments of the disclosure, the plant is transformed with a plasmidthat contains transcription unit sets TUS₁, TUS₂, and TUS₄.

In other embodiments of the disclosure, the plant is transformed with aplasmid that contains transcription unit sets TUS₁, and TUS₃. In otherembodiments of the disclosure, the plant is transformed with a plasmidthat contains transcription unit sets TUS₁, TUS₃, and TUS₄.

In other embodiments of the disclosure, the plant is transformed with aplasmid that contains transcription unit sets TUS₁, and TUS₄. In someembodiments of the disclosure, transgenic plants are prepared bycrossing a first plant that has been transformed with a plasmidcontaining one or more transcription unit sets with a seconduntransformed plant. In other embodiments of the disclosure, transgenicplants are prepared by crossing a first plant that has been transformedwith a plasmid containing one or more but not all transcription unitsets required for micelle formation in vivo with a second plant havingone or more transcription unit sets, wherein at least one of thetranscription unit sets is present in the second plant and not presentin the first plant.

In some embodiments of the disclosure, transgenic plants are prepared bycrossing a first plant that has been transformed with a plasmidcontaining one or more transcription unit sets enabling micelleformation in vivo with a second plant having another trait, such asherbicide resistance or pest resistance.

In some embodiments of the disclosure, transgenic plants are prepared bygrowing progeny generations of a plant that has been transformed with aplasmid containing one or more transcription unit sets enabling micelleformation in vivo. In other embodiments, transgenic plants are preparedby growing progeny generations of a transgenic plant produced bycrossing one or more plants that have been transformed with a plasmidcontaining one or more transcription unit sets enabling micelleformation in vivo.

Further to this example shown in FIG. 2 as an embodiment, the promotersin the four transcription unit sets include the promoters of genescoding for soybean Glycinin1, soybean β-conglycinin1, soybean Glycinin4,soybean Bowman-Birk protease inhibitor, Agrobacterium nopaline synthase,soybean Glycinin5, soybean lectin, and soybean Glycinin3. For clarityand brevity, the promoters of genes coding for soybean Glycinin1 isshown and annotated in FIG. 2 as GY1 PROMOTER. Also for clarity andbrevity, the soybean β-conglycinin1 is shown and annotated in FIG. 2 asCG1 promoter. Further for clarity and brevity, the soybean Glycinin4 isshown and annotated in FIG. 2 as GY4 promoter. Yet further for clarityand brevity, the Bowman-Birk protease inhibitor promoter is shown andannotated in FIG. 2 as D-II promoter. Yet further for clarity andbrevity, the Agrobacterium nopaline synthase is shown and annotated inFIG. 2 as NOS promoter. Yet further for clarity and brevity, the soybeanGlycinin5 is shown and annotated in FIG. 2 as GY5 promoter. Yet furtherfor clarity and brevity, the soybean lectin is shown and annotated inFIG. 2 as LE1 promoter. Yet further for clarity and brevity, the soybeanGlycinin3 is shown and annotated in FIG. 2 as GY3 promoter.

In other embodiments and examples, promoters in one or more of the fourtranscription unit sets include a promoter capable of initiatingtranscription in plant cells whether or not an origin of the promoter isa plant cell. For example, Agrobacterium promoters are functional inplant cells. The promoters capable of initiating transcription in plantcells include promoters obtained from plants, plant viruses and bacteriasuch as Agrobacterium.

As specific examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, or seeds. Such promoters are referred to as“tissue preferred”. Also as specific examples of promoters that initiatetranscription only in certain tissues are referred to as “tissuespecific”. Further as a specific example, a “cell type specific”promoter primarily drives expression in certain cell types in one ormore organs, for example, vascular cells in roots or leaves. Yet furthera specific example, an “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may affect transcription by inducible or repressiblepromoters include anaerobic conditions, or certain chemicals, or thepresence of light. Tissue preferred, tissue specific, cell typespecific, and inducible or repressible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterwhich is active under most conditions.

Returning to this example in FIG. 2 as an embodiment, thetranscriptional terminators in the four transcription unit sets includethe termination sequence of the nopaline synthase gene, shown andannotated in FIG. 2 as NOS terminator. In other embodiments, thetranscriptional terminators in one or more of the four transcriptionunit sets includes transcriptional terminators from the native soybeanGlycinin genes, or any other plant transcriptional terminators.

In this example as an embodiment, the T-DNA used to transform a plantalso includes DNA encoding an origin of replication, a gene conferringantibiotic resistance, a right boundary for the T-DNA, and a leftboundary for the T-DNA, shown and annotated in FIG. 2 as pUC origin,ampicillin resistance, RB, and LB, respectively. In this embodiment, thegene conferring antibiotic resistance is a gene conferring resistance tothe antibiotic ampicillin. In other embodiments, the gene conferringantibiotic resistance is a gene conferring resistance to any otherantibiotic, including kanamycin and chloramphenicol.

Referring now to FIG. 3A, therein is shown an example with additionaldetails from the plant transformation to the post-translationmodification. The plant transformation and the post-translationmodification are also described in FIG. 1. The example depicted in FIG.3A also depicts the recombinant casein protein formation, also describedin FIG. 1. As a specific example, FIG. 3A schematically illustrates thetranscription of casein proteins from genes in TUS₁ as well as posttranscriptional alterations that occur as the proteins move towardstheir subcellular specific destination encoded by the common signalpeptide.

In the example shown in FIG. 3A, the purpose of transcription unit set 1is forming casein micelles in vivo in an embodiment. In this example,the plant transformation depicts a plant transformed using a T-DNAincluding four transcription unit sets shown and annotated in FIG. 3A as4-TUS plasmid. The T-DNA includes transcription unit set 1, shown andabbreviated in FIG. 3A as TUS₁, which includes one transcription unitfor each of the four casein proteins found in a casein micelle, witheach transcription unit including DNA encoding the same plant-derivedsignal peptide, a promoter and a transcriptional terminator as describedin FIG. 2. Upon transcription and translation of TUS₁ in the transgenicplant during the recombinant casein protein formation, the fourrecombinant casein proteins (αS₁-casein, αS₂-casein, β-casein, andκ-casein) are formed in the cytoplasm, each including a signal peptidethat localizes the recombinant protein to a specific tissue, for examplethe secretory pathway and protein storage vacuoles, in the plant cell.In this example, the signal peptide is removed from the recombinantcasein proteins during post-translational modification that occurs inthe endoplasmic reticulum, abbreviated as ER, of the plant cell.

Continuing this example and embodiment for the post-translationmodification, phosphorylation occurs on the recombinant casein proteinsprior to, during, or after migration to a specific tissue. Thephosphorylation is shown in FIG. 3A as circles enclosing the letter “P”that are added to and then attached to each of the recombinant caseinproteins to form phosphorylated casein proteins. The phosphorylatedcasein proteins are then localized to the vacuole where micelle assemblyoccurs in vivo. In some embodiments, proteins encoded by TUS₂transcription units (not shown) are also phosphorylated, glycosylated,or a combination thereof. In other embodiments, the casein proteinsencoded by TUS₄ transcription units (not shown) are also phosphorylatedor glycosylated or both. In other embodiments, no post-translationalmodifications occur to proteins encoded by TUS 1, TUS₂, TUS₃, or TUS₄(not shown). As another example and embodiment, a kinase gene mayoptionally be included to generate a kinase protein that ensuresphosphorylation of the casein proteins encoded by TUS₄ transcriptionunits (not shown).

Referring now to FIG. 3B, therein is shown an example with additionaldetails for the in vivo formation. The in vivo formation is alsodescribed in FIG. 1. As a specific example, FIG. 3B schematicallyillustrates the in vivo formation of recombinant micelles inside a plantcell.

Upon localization to the vacuole, each of the four recombinant caseinproteins assemble with the other recombinant casein proteins to formmicelles in vivo. In this example, the outer layer of the micelle isenriched in recombinant κ-casein shown and abbreviated in FIG. 3B as κ,and the inner matrix of the micelle includes recombinant αS₁-casein andαS₂-casein, shown and abbreviated as αS₁ and αS₂, respectively, in FIG.3B, and β-casein, shown and abbreviated in FIG. 3B as (3.

Referring now to FIG. 3C, therein is shown an example of a schematicillustration of a transcription of proteins which impart herbicideresistance to the transformed plant. FIG. 3C depicts an example of thepurpose of transcription unit set 2 in an embodiment.

In this example, a plant is transformed using a T-DNA including fourtranscription unit sets shown and annotated in FIG. 3C as 4-TUS plasmid.The T-DNA includes transcription unit set 2, shown in FIG. 3C andabbreviated as TUS₂ which includes one transcription unit that includesDNA encoding phosphinothricin acetyltrasnferase that imparts herbicideresistance and allow for selection of transformed cells producingmicelles, shown and abbreviated as AC-PT in FIG. 3C, and a promoter anda transcriptional terminator (not shown).

Referring now to FIG. 3D, therein is shown an example of a schematicillustration of suppression of native seed storage proteins byinterference RNA (RNAi) transcribed by a portion of the plasmid ofFIG. 1. As a specific example, FIG. 3D schematically illustratessuppression of native seed storage proteins by RNAi transcribed by oneor more genes in TUS₃.

FIG. 3D depicts an example of the purpose of transcription unit set 3 inan embodiment. In this example, a plant is transformed using a T-DNAincluding four transcription unit sets, shown and annotated in FIG. 3Das 4-TUS plasmid. The T-DNA includes transcription unit set 3, shown andabbreviated in FIG. 3D as TUS₃, which includes one or more transcriptionunits that yield untranslated RNA molecules that suppress native seedprotein gene translation thereby freeing cellular resources to producemicelles in vivo. Transcription of the DNA in TUS₃ yields RNAi, shownand abbreviated in FIG. 3D as RNAi, that targets messenger RNA of nativeplant proteins or native plant peptides, shown and annotated in FIG. 3Das mRNA NATIVE PROTEIN, and suppresses the expression of those messengerRNAs through messenger RNA degradation such that the recombinant caseinproteins encoded by TUS₁, described in FIG. 1, FIG. 3A, and FIG. 3B, canbe translated at higher quantities, thereby yielding higherconcentrations of micelles in vivo (not shown). In some embodiments, DNAencoding RNAi is operably linked to a constitutive promoter or aninducible promoter (not shown), such as for example a nopaline synthasepromoter or soybean α′ subunit of β-conglycinin, such that thesuppression of native seed protein gene translation by RNAi can beregulated.

Referring now to FIG. 3E and FIG. 3F, therein are shown examples ofschematic illustrations of a transcription of a portion of the plasmidof FIG. 1 and resulting proteins used to alter the intracellularconditions of the plant cell. As specific examples, FIG. 3E and FIG. 3Fschematically illustrate the transcription of TUS₄ genes and theresulting proteins used to alter the conditions in the cytoplasm of thecell.

FIG. 3E depicts an example of the purpose of transcription unit set 4 inan embodiment. In this example, a plant is transformed using a T-DNAincluding four transcription unit sets, shown and annotated in FIG. 3Eas 4-TUS plasmid. The T-DNA includes transcription unit set 4, shown andabbreviated in FIG. 3E as TUS₄, which includes one or more transcriptionunits that encode proteins which increase the concentration ofintracellular minerals, including calcium and phosphate. In thisexample, TUS₄ includes one transcription unit, a TU₄₋₁, that includes apromoter, DNA encoding oxalate decarboxylase, and a transcriptionalterminator (not shown). Transcription and translation of TU₄₋₁ yieldsthe enzyme oxalate decarboxylase, shown and abbreviated in FIG. 3E asOD, that breaks down the calcium oxalate and increases calcium levels inthe plant cell. The increased intracellular calcium enhances theformation of recombinant casein micelles in the plant cell (not shown).

FIG. 3F depicts an example of the purpose of transcription unit set 4 inan embodiment. In this example, a plant is transformed using a T-DNAincluding four transcription unit sets, shown and annotated in FIG. 3Fas 4-TUS plasmid. The T-DNA includes transcription unit set 4, shown andabbreviated in FIG. 3F as TUS₄, which includes one or more transcriptionunits that encode proteins which increase the concentration ofintracellular minerals, including calcium and phosphate. In thisexample, TUS₄ includes one transcription unit, a TU₄₋₂, that includes apromoter, DNA encoding a phytase enzyme, and a transcriptionalterminator (not shown). Transcription and translation of TU₄₋₂ yieldsthe phytase enzyme, shown and abbreviated in FIG. 3F as PE, that breaksdown the phytic acid and increases phosphate levels in the plant cell.The increased intracellular phosphate enhances the formation ofrecombinant casein micelles in the plant cell (not shown).

Referring now to FIG. 3G, therein is shown an example of furtheradditional details of the in vivo formation. The in vivo formation isalso described in FIG. 1, FIG. 3A, and FIG. 3B. As a specific example,FIG. 3G schematically illustrates the in vivo formation of recombinantmicelles inside a plant cell.

In the example shown in FIG. 3G, the in vivo formation of recombinantmicelles inside a plant cell in which the four micellar proteins areproduced by transcription and translation of transcription unit set 1 asdepicted and described in FIG. 3A. The levels of calcium in plant cellvacuoles is increased by the presence of oxalate decarboxylase producedby transcription and translation of transcription unit set 4 as depictedand described in FIG. 3E. In this example, the four casein proteinsencoded by transcription unit 1 are phosphorylated and localized to theplant cell vacuole where the intracellular calcium and the intracellularphosphate enhances the formation of recombinant casein micelles in theplant cell vacuole.

Aspects of the disclosure can be further illustrated by a specificembodiment in which a casein micelle is assembled in vivo from itsconstituent proteins in Arabidopsis thaliana as further described inFIG. 4 and FIG. 5.

Referring now to FIG. 4, therein is shown an example of a schematicillustration of a portion of a plasmid in Arabidopsis. The example shownin FIG. 4 is also described in FIG. 2. As a specific example, FIG. 4schematically illustrates elements of plasmids that encode micellarcomponent proteins. Transcription units depicted are components of TUS₁in Arabidopsis.

The example in FIG. 4 depicts a transcription unit set which can be usedfor creation of casein micelles in vivo in Arabidopsis thaliana. Thetranscription unit set includes one transcription unit for each of thefour casein proteins found in a casein micelle, abbreviated and shown inFIG. 4 as TU₁₋₁, TU₁₋₂, TU₁₋₃, and TU₁₋₄. The transcription unit setabbreviated and shown in FIG. 4 as TUS₁. Each of the four transcriptionunits includes a promoter, a plant-derived N-terminal signal peptide,DNA encoding one of the four proteins found in a casein micelle, and atranscriptional terminator.

Continuing this example, TU₁₋₁ includes a double 35S promoter containingthe tobacco mosaic virus omega leader sequence (SEQ ID NO:29), a signalpeptide from the Arabidopsis CLV3 gene (SEQ ID NO:27), the αS₁-caseincoding sequence codon optimized for expression in Arabidopsis with aC-terminal HDEL peptide for retention in the endoplasmic reticulum (SEQID NO:5), and the nopaline synthase terminator (SEQ ID NO:35), annotatedand shown in FIG. 4 as 2X35S promoter+TMVΩ, signal peptide, αS₁ casein,and NOS terminator, respectively.

Further continuing this example, TU₁₋₂ includes a 35S short promotercontaining a truncated version of the cauliflower mosaic virus promoterand the tobacco mosaic virus omega leader sequence (SEQ ID NO:31), asignal peptide (SEQ ID NO:27), the β-casein coding sequence codonoptimized for expression in Arabidopsis with a C-terminal HDEL peptidefor retention in the endoplasmic reticulum (SEQ ID NO:7), and thenopaline synthase terminator (SEQ ID NO:35), abbreviated and shown inFIG. 4 as 35S SHORT PROMOTER+TMVΩ, SIGNAL PEPTIDE, β-CASEIN, and NOSTERMINATOR, respectively.

Further continuing this example, TU₁₋₃ includes the mannopine synthasepromoter from Agrobacterium tumefaciens (SEQ ID NO:32), a signal peptide(SEQ ID NO:27), the κ-casein coding sequence codon optimized forexpression in Arabidopsis with a C-terminal HDEL peptide for retentionin the endoplasmic reticulum (SEQ ID NO:6), and the nopaline synthaseterminator (SEQ ID NO:35), abbreviated and shown in FIG. 4 as MANNOPINESYNTHASE PROMOTER (A. Tumefaciens), SIGNAL PEPTIDE, κ-CASEIN, and NOSTERMINATOR, respectively.

Further continuing this example, TU₁₋₄ includes the mannopine synthasepromoter from Agrobacterium tumefaciens, a signal peptide (SEQ IDNO:32), the αS₂-casein coding sequence codon optimized for expression inArabidopsis with a C-terminal HDEL peptide for retention in theendoplasmic reticulum (SEQ ID NO:8), and the nopaline synthaseterminator (SEQ ID NO:35), abbreviated and shown in FIG. 4 as MANNOPINESYNTHASE PROMOTER (A. Tumefaciens), SIGNAL PEPTIDE, αS₂ CASEIN, and NOSTERMINATOR, respectively.

Referring now to FIG. 5, therein is shown an example of a schematicillustration of a portion of a plasmid in Arabidopsis for a screenablemarker in plants. As a specific example, FIG. 5 schematicallyillustrates elements of plasmids that provide for a screenable marker inplants. Transcription units depicted are components of TUS₂ inArabidopsis.

The example shown in FIG. 5 depicts a transcription unit set which canbe used to identify plant cells that have been transformed. Thetranscription unit set, abbreviated and shown in FIG. 5 as TUS₂,includes a single transcription unit, abbreviated and shown in FIG. 5 asTU₂₋₁.

Continuing this example for a portion of the plant transformation shownin FIG. 5 as an embodiment, TU₂₋₁ includes the nopaline synthaseconstitutive promoter (SEQ ID NO:28), the enhanced green fluorescenceprotein coding sequence modified to enhance fluorescence brightness andcodon optimized for expression in Arabidopsis (SEQ ID NO:33), and thenopaline synthase terminator (SEQ ID NO:35), abbreviated and shown inFIG. 5 as NOS PROMOTER, EGFP, and NOS TERMINATOR, respectively.

As a specific example, subsequent steps in the plant transformation forcreation of casein micelles in vivo in Arabidopsis thaliana, a plasmidincluding TUS₁ and TUS₂ can be introduced into Arabidopsis thalianacotyledons using Agrobacterium tumefaciens and the FAST transientexpression method. Seedlings are soaked in a solution containingAgrobacterium two days after germination which results in some cotyledoncells being transformed. Transformed Arabidopsis cells can be identifiedas containing the T-DNA by observing fluorescence exhibited by theenhanced green fluorescence protein. Successfully transformedArabidopsis cells display green fluorescence while unsuccessfullytransformed cells show little or no green fluorescence.

Also as a specific example of the in vivo formation of micelles inArabidopsis thaliana as an embodiment, immunogold labeling techniquescan be used to identify the location and morphology of the caseinmicelles formed in vivo. For this example for the in vivo formation ofmicelles as an embodiment, embryonic tissue can be obtained fromArabidopsis thaliana that has been transformed with a plasmid includingTUS 1, and optionally TUS₂, shown in FIG. 4 and FIG. 5, respectively.The embryonic tissue can be treated with casein-specific antibodiesusing immunogold labeling techniques, and imaged with transmissionelectron microscopy to identify the location and morphology of themicelles formed in vivo. In tissue obtained from the transformedArabidopsis thaliana, the casein micelles are visualized asgold-antibody labeled subcellular structures that range in size from 50nm to 600 nm, which is similar to the size of bovine casein micelles. Asa control, no casein micelles are visualized using immunogold labelingtechniques in tissue obtained from untransformed Arabidopsis thaliana.

Continuing this specific example of the in vivo formation of micelles inArabidopsis thaliana as an embodiment, protein extraction and highperformance liquid chromatography (HPLC) analysis can be used toevaluate the protein composition of the casein micelles formed in vivo.In this example for the in vivo formation of micelles as an embodiment,embryonic tissue can be obtained from Arabidopsis thaliana that has beentransformed with a plasmid including TUS₁, and optionally TUS₂, shown inFIG. 4 and FIG. 5, respectively. Proteins extracted from the embryonictissue can be separated using HPLC and detected by ultravioletabsorbance. Proteins extracted from the transformed Arabidopsis thalianatissue and subjected to HPLC analysis show peaks associated with eachfour proteins found in a casein micelle, including αS₁ casein, αS₂casein, β casein, and κ casein, that display retention times similar tothose reported by Bordin et al. for each of the four casein proteinsfound in bovine casein micelles. As a control, proteins extracted fromthe untransformed Arabidopsis thaliana tissue and subjected to HPLCanalysis do not show peaks associated with any of the four caseinproteins.

Further continuing this specific example of the in vivo formation ofmicelles in Arabidopsis thaliana as an embodiment, the amount of eachcasein protein found in micelles formed in vivo can be quantified bymeasuring the area under the peaks produced upon HPLC analysis.Quantification of the peaks produced upon HPLC analysis of proteinsextracted from transformed Arabidopsis thaliana produces measurementsshowing that αS₁ casein is the most abundant, followed by β casein asthe next most abundant, then αS₂ casein and κ casein as the leastabundant casein proteins, which correlates to the relative abundances ofeach of the four casein proteins in bovine casein micelles as previouslyreported in the Handbook of Dairy Foods and Nutrition, Table 1.1.

Aspects of the disclosure can be further illustrated by a specificembodiment in which a casein micelle is assembled in vivo from itsconstituent proteins in soybean and further described in FIG. 6 throughFIG. 9.

Referring now to FIG. 6, therein is shown an example of a schematicillustration of a portion of a plasmid in soybean. As a specificexample, FIG. 6 schematically illustrates elements of plasmids thatencode micellar component proteins. Transcription units depicted arecomponents of TUS₁ in soybean.

In this example, FIG. 6 depicts a transcription unit set which can beused for creation of casein micelles in vivo in soybean. Thetranscription unit set includes one transcription unit for each of thefour casein proteins found in a casein micelle, abbreviated and shown inFIG. 4 as TU₁₋₁, TU₁₋₂, TU₁₋₃, and TU₁₋₄. The first transcription unitset is abbreviated and shown in FIG. 6 as TUS 1. Each of the fourtranscription units includes a promoter, a plant-derived N-terminalsignal peptide, DNA encoding one of the four proteins found in a caseinmicelle, and a transcriptional terminator.

Continuing this example for a portion of the plant transformation shownin FIG. 6 as an embodiment, TU₁₋₁ includes a promoter from the soybeanglycinin GY1 gene (SEQ ID NO:13), a signal peptide (SEQ ID NO:26), theαS₁ casein coding sequence codon optimized for expression in soybean(SEQ ID NO:1), and the nopaline synthase terminator (SEQ ID NO:35),abbreviated and shown in FIG. 6 as GY1 PROMOTER, SIGNAL PEPTIDE, αS₁CASEIN, and NOS TERMINATOR, respectively.

Further continuing this example for a portion of the planttransformation shown in FIG. 6 as an embodiment, TU₁₋₂ includes thepromoter from the soybean CG1 gene (SEQ ID NO:14), a signal peptide (SEQID NO:26), the β casein coding sequence codon optimized for expressionin soybean (SEQ ID NO:3), and the nopaline synthase terminator (SEQ IDNO:35), abbreviated and shown in FIG. 6 as CG1 PROMOTER, SIGNAL PEPTIDE,(3 CASEIN, and NOS TERMINATOR, respectively.

Further continuing this example for a portion of the planttransformation shown in FIG. 6 as an embodiment, TU₁₋₃ includes thepromoter from the soybean glycinin GY4 gene (SEQ ID NO:15), a signalpeptide (SEQ ID NO:26), the κ casein coding sequence codon optimized forexpression in soybean (SEQ ID NO:2), and the nopaline synthaseterminator (SEQ ID NO:35), abbreviated and shown in FIG. 6 as GY4PROMOTER, SIGNAL PEPTIDE, κ CASEIN, and NOS TERMINATOR, respectively.

Further continuing this example for a portion of the planttransformation shown in FIG. 6 as an embodiment, TU₁₋₄ includes thepromoter from the soybean D-II Bowman-Birk proteinase isoinhibitor gene(SEQ ID NO:16), a signal peptide (SEQ ID NO:26), the 1362 casein codingsequence codon optimized for expression in soybean (SEQ ID NO:4), andthe nopaline synthase terminator (SEQ ID NO:35), abbreviated and shownin FIG. 6 as D-II PROMOTER, SIGNAL PEPTIDE, αS₂ CASEIN, and NOSTERMINATOR, respectively.

Referring now to FIG. 7, therein is shown an example of a schematicillustration of a portion of a plasmid in soybean for herbicideresistance in plants. As a specific example, FIG. 7 schematicallyillustrates elements of plasmids that provide for herbicide resistancein plants. Transcription units depicted are components of TUS₂ insoybean.

FIG. 7 is an example of a portion of the plant transformation thatdepicts a transcription unit set which can be used to select for plantcells that have been transformed. The transcription unit set abbreviatedand shown in FIG. 7 as TUS₂ includes a single transcription unitabbreviated and shown in FIG. 7 as TU₂₋₁.

Continuing this example for a portion of the plant transformation shownin FIG. 7 as an embodiment, TU₂₋₁ includes nopaline synthase promoter(SEQ ID NO:28), the phosphinothricin acetyltransferase coding sequencecodon optimized for expression in soybean (SEQ ID NO:34) which confersresistance to the herbicide glufosinate, and the nopaline synthaseterminator (SEQ ID NO:35), abbreviated and shown in FIG. 7 as NOSPROMOTER, PHOSPHINOTHRICIN ACETYLTRANSFERASE, and NOS TERMINATOR,respectively.

Referring now to FIG. 8, therein is shown an example of a schematicillustration of a portion of a plasmid in soybean for suppression ofnative seed storage proteins in plants. As a specific example, FIG. 8schematically illustrates elements of plasmids that provide forsuppression of native seed storage proteins in plants. Transcriptionunits depicted are components of TUS₃ in soybean.

FIG. 8 is an example of a portion of the plant transformation thatdepicts a transcription unit set which can be used for enhancing thecreation of casein micelles in vivo in soybean. The third transcriptionunit set abbreviated and shown in FIG. 8 as TUS₃ includes twotranscription units abbreviated and shown in FIG. 8 as TU₃₋₁ and TU₃₋₂.The transcription of TU₃₋₁ and TU₃₋₂ produces RNA with a hairpinstructure where the arms are homologous to a portion of a native soybeangene or gene family and are sufficient to cause down regulation of thosenative genes or gene families (not shown).

Continuing this example for a portion of the plant transformation shownin FIG. 8 as an embodiment, TU₃₋₁ includes a promoter from the soybeanglycinin GY4 gene (SEQ ID NO:15), a portion of the soybean glycinin GY1coding sequence that is lacking a start codon and is highly homologousamong the glycinin gene family (SEQ ID NO:24), the potato IV2 intron(SEQ ID NO:25), the antisense of the soybean glycinin GY1 sequence (SEQID NO:17), and the nopaline synthase terminator (SEQ ID NO:35),abbreviated and shown in FIG. 8 as GY4 PROMOTER, GY1 SENSE, IV2 INTRON,GY1 ANTISENSE, and NOS TERMINATOR, respectively.

Further continuing this example for a portion of the planttransformation shown in FIG. 8 as an embodiment, TU₃₋₂ includes apromoter from the soybean glycinin GY5 gene (SEQ ID NO:18), a portion ofthe soybean β-conglycinin 1 coding sequence that is lacking a startcodon and is highly homologous among the β-conglycinin gene family (SEQID NO:19), the potato IV2 intron (SEQ ID NO:25), the antisense of thesoybean β-conglycinin 1 sequence (SEQ ID NO:20), and the nopalinesynthase terminator (SEQ ID NO:35), abbreviated and shown in FIG. 8 asGY5 PROMOTER, CG1 SENSE, IV2 INTRON, CG1 ANTISENSE, and NOS TERMINATOR,respectively.

Referring now to FIG. 9, therein is shown an example of a schematicillustration of a portion of a plasmid in soybean for regulatingcytoplasmic concentrations of minerals which can enhance micelleformation. As a specific example, FIG. 9 schematically illustrateselements of plasmids that regulate cytoplasmic concentrations ofminerals which can enhance micelle formation. Transcription unitsdepicted are components of TUS₄ in soybean.

FIG. 9 is an example of a portion of the plant transformation thatdepicts a transcription unit set which can be used for enhancing thecreation of casein micelles in vivo in soybean. The fourth transcriptionunit set abbreviated and shown in FIG. 9 as TUS₄ includes twotranscription units abbreviated and shown in FIG. 9 as TU₄₋₁ and TU₄₋₂.Proteins encoded by TU₄₋₁ and TU₄₋₂ alter the intracellular environmentin a manner that optimizes the formation of micelles in vivo.

Continuing this example for a portion of the plant transformation shownin FIG. 9 as an embodiment, TU₄₋₁ includes a promoter from the soybeanLE1 gene (SEQ ID NO:23), a coding sequence for oxalate decarboxylasefrom Flammulina velutipes codon optimized for expression in soybean (SEQID NO:12), and nopaline synthase terminator (SEQ ID NO:35), abbreviatedand shown in FIG. 9 as LE1 PROMOTER, OXALATE DECARBOXYLASE CDS, and NOSTERMINATOR, respectively.

Further continuing this example for a portion of the planttransformation shown in FIG. 9 as an embodiment, TU₄₋₂ includes theglycinin GY3 promoter (SEQ ID NO:30), the coding sequence for a soybeanphytase enzyme (SEQ ID NO:11), and the nopaline synthase terminator (SEQID NO:35), abbreviated and shown in FIG. 9 as GY3 PROMOTER, PHYTASE, andNOS TERMINATOR, respectively.

In this example, subsequent steps in the plant transformation forcreation of casein micelles in vivo in soybean, a plasmid includingTUS₁, TUS₂, and optionally TUS₃, and optionally TUS₄, shown in FIG. 6,FIG. 7, FIG. 8, and FIG. 9, respectively, can be introduced into soybeancallus using standard biolistic transformation methods. Transformedsoybean plants can be selected on a medium containing glufosinateherbicide, and the genomes of transformed soybean plants can be screenedfor insertion of the plasmid using standard PCR mapping methods.Transformed soybean plants including TUS₁, TUS₂, and optionally TUS₃,and optionally TUS₄ in their genome can be transferred to a greenhousefor seed production.

In the example of the in vivo formation of micelles in soybean as anembodiment, immunogold labeling techniques can be used to identify thelocation and morphology of the casein micelles formed in vivo. As itrelates to this example for the in vivo formation of micelles as anembodiment, tissue can be obtained from soybean plants that have beentransformed with a plasmid including TUS₁, TUS₂, and optionally TUS₃,and optionally TUS₄, shown in FIG. 6, FIG. 7, FIG. 8, and FIG. 9,respectively. The tissue can be treated with casein-specific antibodiesusing standard immunogold labeling techniques, and imaged withtransmission electron microscopy to identify the location and morphologyof the micelles formed in vivo. In tissue obtained from the transformedsoybean plants, the casein micelles are visualized as gold-antibodylabeled subcellular structures that range in size from 50 nm to 600 nm,which is similar to the size of bovine casein micelles. As a control, nocasein micelles are visualized using immunogold labeling techniques intissue obtained from untransformed soybean plants.

Continuing this example of the in vivo formation of micelles in soybeanas an embodiment, protein extraction and polyacrylamide gelelectrophoresis analysis can be used to evaluate the protein compositionof the casein micelles formed in vivo. For this example for the in vivoformation of micelles as an embodiment, tissue can be obtained fromsoybean plants that have been transformed with a plasmid including TUS₁,TUS₂, and optionally TUS₃, and optionally TUS₄, shown in FIG. 6, FIG. 7,FIG. 8, and FIG. 9, respectively. Proteins extracted from thetransformed soybean plant tissue and subjected to polyacrylamide gelelectrophoresis analysis show bands on the polyacrylamide gelcorresponding in size to each of the four casein proteins found in acasein micelle, including αS₁ casein, αS₂ casein, 13 casein, and κcasein. As a control, proteins extracted from untransformed soybeanplant tissue and subjected to polyacrylamide gel electrophoresisanalysis do not show bands on the polyacrylamide gel corresponding tothe four casein proteins.

Further continuing this example of the in vivo formation of micelles insoybean as an embodiment, protein extraction and HPLC analysis can beused to evaluate the protein composition of the casein micelles formedin vivo. For this example for the in vivo formation of micelles as anembodiment, tissue can be obtained from soybean plants that have beentransformed with a plasmid including TUS₁, TUS₂, and optionally TUS₃,and optionally TUS₄, shown in FIG. 6, FIG. 7, FIG. 8, and FIG. 9,respectively. Proteins extracted from the transformed soybean planttissue can be separated using HPLC and detected by ultravioletabsorbance. Proteins extracted from the transformed soybean plant tissueand subjected to HPLC analysis show peaks associated with each fourproteins found in a casein micelle, including αS₁ casein, αS₂ casein, βcasein, and κ casein, that display retention times similar to thosereported by Bordin et al. for each of the four casein proteins found inbovine casein micelles. As a control, proteins extracted from theuntransformed soybean plant tissue and subjected to HPLC analysis do notshow peaks associated with the four casein proteins.

Further continuing this example of the in vivo formation of micelles insoybean as an embodiment, the amount of each casein protein found inmicelles formed in vivo can be quantified by measuring the area underthe peaks produced upon HPLC analysis. Quantification of the peaksproduced upon HPLC analysis of proteins extracted from transformedsoybean plant tissue produces measurements showing that αS₁ casein isthe most abundant, followed by β casein as the next most abundant, thenαS₂ casein and κ casein as the least abundant casein proteins, whichcorrelates to the relative abundances of each of the four caseinproteins in bovine casein micelles as previously reported in theHandbook of Dairy Foods and Nutrition, Table 1.1.

Further continuing this example of the in vivo formation of micelles insoybean as an embodiment, RNA analysis can be used to evaluate thesuppression of native soybean seed genes during the formation of caseinmicelles in vivo. For this example for the in vivo formation of micellesas an embodiment, soybean plants that have been transformed with aplasmid including TUS₁, TUS₂, TUS₃, and optionally TUS₄, shown in FIG.6, FIG. 7, FIG. 8, and FIG. 9, respectively, can be grown to theflowering stage in a greenhouse and soybean embryos removed from theflowering seed pods at 35 days using standard dissection techniques. Theexpression levels of native soybean seed genes can be analyzed usingstandard techniques for RNA extraction and sequencing. RNA analysis ofthe embryos from transformed soybean plants show at least a 10%reduction in the expression of one or more of the native soybean seedgenes, including genes in the glycinin family (Glyma.03g163500,Glyma.19g164900, Glyma.10g037100, Glyma.13g123500, Glyma.19g164800) andgenes in the β-conglycinin family (Glyma.10g246300, Glyma.20g148400,Glyma.20g148300, Glyma.20g146200, Glyma.20g148200, Glyma.10g246500,Glyma.10g028300, Glyma.02g145700). As a control, RNA analysis of embryosfrom untransformed soybean plants do not show a reduction in theexpression of native soybean seed genes.

Further continuing this example of the in vivo formation of micelles insoybean as an embodiment, commercially available assays and X-rayfluorescence techniques can be used to evaluate calcium oxalate levelsduring the formation of casein micelles in vivo. As it relates to thisexample for the in vivo formation of micelles as an embodiment, soybeanplants that have been transformed with a plasmid including TUS₁, TUS₂,and optionally TUS₃, and TUS₄, shown in FIG. 6, FIG. 7, FIG. 8, and FIG.9, respectively, can be grown to the flowering stage in a greenhouse andsoybean embryos removed from the flowering seed pods at 27 days usingstandard dissection techniques. The oxalate concentration can bemeasured using commercially available assays, and the calciumconcentration can be measured using X-ray fluorescence. Embryos fromtransformed soybean plants show at least a 5% reduction in oxalateconcentration and at least a 4% increase in calcium concentration ascompared to control embryos from untransformed soybean plants,indicating that embryos from transformed soybean plants have at least 4%more available calcium compared to embryos from untransformed soybeanplants.

Further continuing this example of the in vivo formation of micelles insoybean as an embodiment, commercially available assays can be used toevaluate phosphate levels during the formation of casein micelles invivo. As it relates to this example for the in vivo formation ofmicelles as an embodiment, soybean plants that have been transformedwith a plasmid including TUS₁, TUS₂, and optionally TUS₃, and TUS₄,shown in FIG. 6, FIG. 7, FIG. 8, and FIG. 9., respectively, can be grownto the flowering stage in a greenhouse and soybean embryos removed fromthe flowering seed pods at 27 days using standard dissection techniques.Embryos can be ground with a mortar and pestle, sonicated andcentrifuged to produce a supernatant that can be tested for phosphataselevels using commercially available assays. Embryos from transformedsoybean plants show at least a 5% increase in phosphatase levels ascompared to control embryos from untransformed soybean plants,indicating that embryos from transformed soybean plants have at least 5%more available phosphate compared to embryos from untransformed soybeanplants.

Aspects of the disclosure can be further illustrated by a specificembodiment in which micelles produced in vivo are purified as furtherdescribed in FIG. 10.

Referring now to FIG. 10, therein is shown an example of a flow for thepurification of micelles formed in vivo in soybean. Also, the flow inFIG. 10 is an example of isolating a recombinant micelle. Further inthis example, FIG. 10 depicts a process where casein micelles producedin soybeans are purified from the plant tissue in a way that themicelles are still functional after the purification. The input materialfor the purification process is dried soybeans harvested from plantsthat have been transformed with a plasmid containing all fourtranscription unit sets, TUS₁, TUS₂, TUS₃, and TUS₄, described in FIG.6, FIG. 7, FIG. 8, and FIG. 9, respectively. The input material for thepurification process is shown in FIG. 10 and depicted as a rectangleenclosing the word “SOYBEAN”.

Continuing this example of the purification of micelles formed in vivoin soybean as an embodiment, the hulls are removed from the driedsoybeans in a series of steps including cleaning, cracking, andaspiration, shown in FIG. 10 and depicted as rectangles enclosing thewords “CLEANING”, “CRACKING” and “ASPIRATION”. In this embodiment, thehulls do not contain useful amounts of casein micelles and arediscarded, shown in FIG. 10 and depicted as an arrow pointing to arectangle enclosing the word “HULLS”.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the remaining material is flaked toincrease the surface area and allow for faster aqueous or solventinfiltrations. The resulting flaked material is shown in FIG. 10 anddepicted as a rectangle enclosing the words “FULL FAT FLAKES”.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the flaked material is thendefatted with hexane using standard defatting equipment and solventextraction techniques, shown in FIG. 10 and depicted as a rectangleenclosing the words “SOLVENT EXTRACTION”. Defatting can occur using anystandard hexane based solvent, followed by desolventizing using flash orvapor-based processes. The resulting oil is removed, shown in FIG. 10and depicted as an arrow pointing to a rectangle enclosing the words“CRUDE OIL”, leaving behind the defatted flakes, shown in FIG. 10 anddepicted as an arrow pointing to a rectangle enclosing the words“DEFATTED SOY FLAKES”.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the defatted flakes are then mixedwith water and wet milled, shown in FIG. 10 and depicted as an arrowpointing to a rectangle enclosing the words “H2O WET MILLING”. Themilling process pulverizes the defatted flakes which releases the caseinmicelles and allows the micelles to come into contact with an aqueousmedium. In addition to the milling process, the defatted flakes are alsovigorously agitated to assist in the release of casein micelles into thewater, shown in FIG. 10 and depicted as an arrow pointing to a rectangleenclosing the word “AGITATION”. The milling process and vigorousagitation of the defatted flakes yields a slurry where soybean materialhas been finely ground and many of the casein micelles have beenreleased into suspension in the water (not shown). Additionally, manyother proteins and carbohydrates are also dissolved in the water (notshown). In some embodiments, wet milling is done using perforated discor colloid continuous flow mills.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the slurry is fed through a seriesof mesh screens to remove larger particles from the casein micelles,shown in FIG. 10 and depicted as an arrow pointing to a rectangleenclosing the word “FILTRATION”. In this embodiment, the slurry is firstpassed through a screen with 5 mm sieve openings (not shown), and thenis passed through a screen with 0.5 mm sieve openings (not shown). Thematerial trapped by the screens is discarded, shown in FIG. 10 anddepicted as an arrow pointing to a rectangle enclosing the words“ORGANIC MATERIAL”.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the remaining material in theslurry that passed through both screens is then sonicated to break upaggregates of casein micelles such that the majority of micelles are notcontacting other micelles, shown in FIG. 10 and depicted as an arrowpointing to a rectangle enclosing the word “SONICATION”. In someembodiments, continuous flow sonication with multiple sonicators inparallel are used to maximize flow rates.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, after sonication the slurry ispassed through a 2 μm microfiltration unit to eliminate larger particleswhile allowing casein micelles to pass through, shown in FIG. 10 anddepicted as an arrow pointing to a rectangle enclosing the word“MICROFILTRATION”. The material trapped by the microfiltration unit isdiscarded, shown in FIG. 10 and depicted as an arrow pointing to arectangle enclosing the words “LARGE PARTICLES”. The remaining materialthat passed through the microfiltration unit is largely composed ofcasein micelles as well as dissolved proteins, salts and carbohydrates(not shown).

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the material that passed throughthe microfiltration unit is then processed with an ultrafiltration unitthat allows dissolved molecules lower than 100 nm in diameter to passthrough while retaining casein micelles, shown in FIG. 10 and depictedas an arrow pointing to a rectangle enclosing the words“ULTRAFILTRATION”. In some embodiments, continuous flow ultrafiltrationwith multiple filters in parallel are used to maximize flow rates. Thesoluble proteins, salts and minerals that passed through theultrafiltration unit are discarded, shown in FIG. 10 and depicted as anarrow pointing to a rectangle enclosing the words “SOL. PROTEINS, SALTS,MINERALS”.

Further continuing this example of the purification of micelles formedin vivo in soybean as an embodiment, the final output from this processis an aqueous liquid where the most common component after water iscasein micelles, shown in FIG. 10 and depicted as an arrow pointing to arectangle enclosing the words “CONCENTRATED MICELLES”. These micelles(not shown) retain their shape and function such that they can be usedin downstream processes such as in making synthetic milk or cheese.

As additional examples for FIG. 10, a method of isolating recombinantmicelles from a seed of a plant produced can include cleaning, anddeshelling or dehulling seeds, flaking cleaned seeds to 0.005-0.02 inchthickness, solvent extraction of oil from the flake, desolventizing theflake without cooking and collecting the defatted, clean flake,separating micelles into an aqueous slurry by hydrating, agitating andwet milling the flake, passing the slurry through a series of meshscreens to remove particulate above 0.5 mm in size and collecting thepermeate, sonication of the permeate from previous step, microfiltrationof the product from previous step to remove particulate above 2 um insize, ultrafiltration of the permeate from previous step using a devicethat allows particles >100 nm in diameter to pass through in theultrafiltration permeate, collecting the retentate of previous stepwhich contains concentrated recombinant micelles.

Continuing with this example, the method of isolating recombinantmicelles from a seed further includes centrifuging the retentate of aprevious step to separate the micelles from the remainder of theretentate. Also the method continues from the ultrafiltration step topassing the slurry through an ultrafiltration device and collecting apermeate containing protein and other molecules and a retentatecontaining micelles and thereafter adding a diafiltration fluid to theretentate at substantially the same rate that the permeate is collectedand passing said retentate through the ultrafiltration device. Yetfurther the method continues where the seed is milled from at least oneplant selected from the group of plants consisting of maize, rice,sorghum, cowpeas, soybeans, cassava, coyam, sesame, peanuts, peas,cotton and yams.

The resulting method, process, apparatus, device, product, and system iscost-effective, highly versatile, and accurate, and can be implementedby adapting components for ready, efficient, and economicalmanufacturing, application, production, and utilization. Anotherimportant aspect of an embodiment of the present disclosure is that itvaluably supports and services the historical trend of reducing costs,simplifying systems, and increasing yield.

These and other valuable aspects of the embodiments of the presentdisclosure consequently further the state of the technology to at leastthe next level. While the disclosure has been described in conjunctionwith a specific best mode, it is to be understood that manyalternatives, modifications, and variations will be apparent to thoseskilled in the art in light of the descriptions herein. Accordingly, itis intended to embrace all such alternatives, modifications, andvariations that fall within the scope of the included claims. Allmatters set forth herein or shown in the accompanying drawings are to beinterpreted in an illustrative and non-limiting sense.

What is claimed is: 1-20. (canceled)
 21. A method of in vivo assembly ofa recombinant micelle, comprising: co-expressing at least two ruminantcasein proteins in a plant cell; wherein the at least two ruminantcasein proteins comprise a κ-casein and at least one of an αS1-casein,an αS2-casein, or a β-casein; and wherein the κ-casein and at least oneof the αS1-casein, the αS2-casein, or the β-casein form the recombinantmicelle in the plant cell.
 22. The method in claim 21, wherein the atleast two ruminant casein proteins are bovine casein proteins.
 23. Themethod in claim 21, wherein co-expressing the at least two ruminantcasein proteins in the plant cell comprises introducing a plasmid intothe plant cell, wherein the plasmid comprises deoxyribonucleic acid(DNA) encoding ribonucleic acid (RNA) for the at least two ruminantcasein proteins.
 24. A recombinant micelle made using the method inclaim
 21. 25. A food product comprising the recombinant micelle in claim24.
 26. The food product in claim 25, wherein the food product is milk.27. The food product in claim 26, wherein the milk is synthetic milk.28. The food product in claim 25, wherein the food product is cheese.29. The food product in claim 28, wherein the cheese is syntheticcheese.
 30. A method of in vivo assembly of a recombinant micellecomprising: introducing a plasmid into a plant cell, wherein the plasmidcomprises 1) a first deoxyribonucleic acid (DNA) sequence encodingribonucleic acid (RNA) for a ruminant κ-casein; and 2) a seconddeoxyribonucleic acid (DNA) sequence encoding ribonucleic acid (RNA) forat least one of a ruminant αS1-casein, a ruminant αS2-casein, or aruminant β-casein; wherein the recombinant micelle is formed andcomprise: 1) an outer layer comprising the ruminant κ-casein, and 2) aninner matrix comprising at least one of the ruminant αS1-casein, theruminant αS2-casein, or the ruminant β-casein.
 31. A method of in vivoassembly of one or more recombinant micelles comprising: introducing atleast one plasmid into a plant cell, wherein the at least one plasmidcomprises a first deoxyribonucleic acid (DNA) sequence encodingribonucleic acid (RNA) for a ruminant κ-casein; and a seconddeoxyribonucleic acid (DNA) sequence encoding ribonucleic acid (RNA) forat least one of a ruminant αS1-casein, a ruminant αS2-casein, or aruminant β-casein; wherein the first and second deoxyribonucleic acid(DNA) sequences are transcribed and translated to produce one or moreproteins comprising the ruminant κ-casein and at least one of a ruminantαS1-casein, a ruminant αS2-casein, or a ruminant β-casein; and whereinthe one or more proteins form one or more recombinant micelles in theplant cell.
 32. A recombinant micelle, comprising at least two ruminantcasein proteins, wherein the at least two ruminant casein proteinscomprise a κ-casein and at least one of an αS1-casein, an αS2-casein, ora β-casein, and wherein the recombinant micelle is assembled in a plantcell by co-expressing the at least two ruminant casein proteins in theplant cell.
 33. The recombinant micelle in claim 32, whereinco-expressing the at least two ruminant casein proteins in the plantcell comprises introducing a plasmid into the plant cell, wherein theplasmid comprises deoxyribonucleic acid (DNA) encoding ribonucleic acid(RNA) for the at least two ruminant casein proteins.
 34. The recombinantmicelle in claim 32, wherein the at least two ruminant casein proteinsare bovine casein proteins.
 35. A food product comprising therecombinant micelle in claim
 32. 36. The food product in claim 35,wherein the food product is milk.
 37. The food product in claim 34,wherein the milk is synthetic milk.
 38. The food product in claim 35,wherein the food product is cheese.
 39. The food product in claim 36,wherein the cheese is synthetic cheese.
 40. A plant cell co-expressingat least two ruminant casein proteins, wherein the at least two ruminantcasein proteins comprise a κ-casein and at least one of an αS1-casein,an αS2-casein, or a β-casein; and wherein the κ-casein and at least oneof the αS1-casein, the αS2-casein, or the β-casein form a recombinantmicelle in the plant cell.
 41. The plant cell in claim 40, whereinco-expressing the at least two ruminant casein proteins in the plantcell comprises introducing a plasmid into the plant cell, wherein theplasmid comprises deoxyribonucleic acid (DNA) encoding ribonucleic acid(RNA) for the at least two ruminant casein proteins.
 42. A plantcomprising the plant cell in claim
 40. 43. The plant in claim 42,wherein the plant is at least one of maize, rice, sorghum, cowpea,soybean, cassava, coyam, sesame, peanut, peas, cotton, or yam.